Allele-Specific rpoB PCR Assays for Detection of Rifampin-Resistant Mycobacterium tuberculosis in Sputum Smears

Abstract

We describe an allele-specific PCR assay to detect mutations in three codons of the rpoB gene (516, 526, and 531) in Mycobacterium tuberculosis strains; mutations in these codons are reported to account for majority of M. tuberculosis clinical isolates resistant to rifampin (RIF), a marker of multidrug-resistant tuberculosis (MDR-TB). Three different allele-specific PCRs are carried out either directly with purified DNA (single-step multiplex allele-specific PCR), or with preamplified rpoB fragment (nested allele-specific PCR [NAS-PCR]). The method was optimized and validated following analysis of 36 strains with known rpoB sequence. A retrospective analysis of the 287 DNA preparations from epidemiologically unlinked RIF-resistant clinical strains from Russia, collected from 1996 to 2002, revealed that 247 (86.1%) of them harbored a mutation in one of the targeted rpoB codons. A prospective study of microscopy-positive consecutive sputum samples from new and chronic TB patients validated the method for direct analysis of DNA extracted from sputum smears. The potential of the NAS-PCR to control for false-negative results due to lack of amplification was proven especially useful in the study of these samples. The developed rpoB-PCR assay can be used in clinical laboratories to detect RIF-resistant and hence MDR M. tuberculosis in the regions with high burdens of the MDR-TB.The spread of multidrug-resistant tuberculosis (MDR-TB) has increased worldwide and reached epidemic proportions in many countries. MDR Mycobacterium tuberculosis strains are considered those resistant to at least rifampin (RIF) and isoniazid. RIF is a key component of the World Health Organization DOTS (directly observed therapy, short course) regimen: because RIF monoresistance is extremely rare and development of isoniazid resistance usually precedes that to RIF, resistance to the latter is considered to be the MDR marker (8). It has been shown in many studies that RIF resistance in up to 95 to 98% of resistant strains is caused by mutations in the rpoB gene encoding the RNA polymerase ␤-subunit (20, 21). These mutations are generally described in the short 81-bp region in rpoB (RIF resistance-determining region: codons 507 to 533 [21]). In addition, RIF resistance may be caused by mutations in other parts of rpoB outside the hot spot region, such as codon 176 (146 [21]) in the N-terminal part (9) and codons 541 and 553 (18). Methods used so far to detect rpoB mutations associated with RIF-resistance were direct DNA sequencing, PCR-single-strand conformation polymorphism, heteroduplex mobility, dot spot, RNA-RNA mismatch, and some other assays (reviewed in references 4 and 25). Previously, we have described a multiplex allele-specific PCR (MAS-PCR) assay based on standard PCR and agarose gel electrophoresis to detect mutations in katG315 (15) and embB306 (14) in M. tuberculosis strains. A similar assay to detect rpsL43, rpoB531, and katG315 mutations was very recently published by Victor et al. (24). The MAS-PCR method in our design (14, 15) ...

Mutations leading to drug-resistant Mycobacterium tuberculosis infection in Ukraine

“…The method targets mutation in codon 315 katG gene with three primers. MAS-PCR was also used in the study of the mutation responsible for rifampicin resistance [4]. The method revealed mutations at codons 516, 526, 531 in the rpoB gene with use of three inner direct primers and one reverse primer.…”

“…The total included 224 cultures of M. tuberculosis; 107 isolates of M. tuberculosis obtained from patients who were treated in that hospital were studied with multiplex allele-specific PCR (MAS-PCR) [3,4]. The DNA extraction was performed in the same laboratory.…”

“…During last two decades, certain mutations in M. tuberculosis genotype that cause drug resistance have been revealed. In the scientific literature, molecular methods are described for detection of mutations in the rpoB and katG genes that lead, respectively, to rifampicin and isoniazid resistance [3,4]. The main advantages of the molecular methods are their high sensitivity and rapidity in obtaining results; however, these methods are not used routinely in Ukraine.…”

Drug-Resistance inMycobacterium Tuberculosis:Molecular Basis and Genotypic Detection

“…the inherent sensitivity limit of genotypic methods to detect RiF, inh, and eMB resistance is explained by the fact that a certain number of isolates have other resistance mechanisms untargeted by these methods -up to 10-15%% in some rpoB studies (4,35,36,37,44). therefore the molecular methods cannot completely substitute culture-based phenotypic tests but may successfully complement them.…”

“…it should be noted that certain resistance linked mutations (katG315 AGc>Acc and rpsL43 AAG>AGG) can be easily detected by the specific PCR-RFLP or allelespecific PCR methodologies (7,33,34,35,36,63). However, such methods targeting alterations in the short gene regions (4-6 bp of the cleavage site) are ineffective for mutations in rpoB, mabA-inhA, ahpC, rpsL88, and rrs, that are scattered throughout more extended regions and/or do not concern actual or potential sites of known restriction endonucleases.…”